Name: milk collection in the rat using capillary tubes and estimation of milk fat content by creamatocrit
Uploaded: 2015-12-16T15:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Milk Collection in the Rat Using Capillary Tubes and Estimation of Milk Fat Content by Creamatocrit at JoVE.com

This works was supported through grants from the Natural Sciences and Engineering Research Council of Canada (RGPIN 238382-2011) and Canadian Institutes of Health Research (MOP115076). Heather Paul was supported by a Natural Sciences and Engineering Research Council of Canada Postgraduate Scholarship and an Alberta Innovates Health Solutions scholarship. Megan Hallam was supported by a Natural Sciences and Engineering Research Council Postgraduate Scholarship, a Frederick Banting and Charles Best Canada Graduate Scholarship, and an Alberta Children&#39;s Hospital Research Institute Training Award in Genetics, Child Development, and Health.

This protocol was approved by the University of Calgary Animal Care Committee and conformed to the Guide for the Care and Use of Laboratory Animals.
1. Separate Dam from Offspring
<ol>
	<li>Separate the dam from her offspring for a minimum of 5 min prior to milking12. 
	NOTE: The dam can be milked up to 5-6 hr after separation1,6,13, however periods of separation longer than 4 hr may alter milk composition14. While separation time does not appear to impact milk collection volume12, it is advised that a consistent separation time be maintained throughout the study. Milk composition may change throughout lactation15, therefore attempts should be made to keep the day of milk collection consistent. Maximum milk production is suggested to occur on lactation day 1412.</li>
	<li>Using a warming chamber, ensure the pups are able to maintain proper body temperature without the presence of their mother for the duration of the milking procedure. 
	NOTE: In the study presented below, milking was performed at weaning, when the dams were approximately 22 weeks old and the offspring 21 days old, therefore no warming chamber was used.</li>
</ol>
2. Set-up and Preparation
<ol>
	<li>Collect all materials required for the milking procedure. 
	NOTE: All materials can be found in the materials and equipment table.</li>
	<li>Place a heating pad on the bench where milking will take place and cover the pad with an absorbent bench under-pad.</li>
	<li>Set up the anesthetic system. Ensure that the system has sufficient oxygen and isoflurane prior to beginning. Attach the anesthetic mask that will be used for initial anesthesia induction to the machine. Place the mask that will be used for anesthesia maintenance nearby if different than the initial anesthetic mask.</li>
	<li>Attach a 25 G needle to a 1 ml syringe for oxytocin injection using aseptic technique.</li>
	<li>Turn on the heating pad so that maternal body temperature is maintained during the milking procedure.&#160;NOTE: Monitor the temperature of the heating pad to ensure the pad does not become too hot and cause burns. Alternatively, use a heat source, such as a heated surgical table, that can be set to a specific temperature.&#160;</li>
</ol>
3. Anesthetize the Dam Using Isoflurane
<ol>
	<li>Open the oxygen tank and turn the flow to 1 L (1,000 cc) per min. Turn on the flow of isoflurane and set to 5%. CAUTION: Avoid direct inhalation of anesthetic and prevent accumulation of anesthetic vapors.</li>
	<li>Anesthetize the dam.</li>
	<li>Switch over to the maintenance mask if required, placing the dam supine on the absorbent bench pad. Confirm anesthetization by lack of pedal reflex.</li>
	<li>Reduce the flow of isoflurane to 2-3% for maintenance of anesthesia. Continually monitor dam throughout the procedure to ensure depression of respiration does not occur.&#160;NOTE: Once under anesthesia, the dam&#8217;s eyes should be protected using a sterile eye lubricant to prevent the eyes from drying out or becoming scratched.&#160;</li>
</ol>
4. Oxytocin Injection
<ol>
	<li>Ensure the oxytocin (20 USP Units/ml) has not passed its expiry date. Disinfect the vial of oxytocin with a sterile alcohol wipe/alcohol cleansing pad.</li>
	<li>Using aseptic technique, draw up 2 IU (0.1 ml) of oxytocin into the syringe. Use a new needle and syringe for each dam that will be milked. 
	NOTE: Oxytocin doses generally range from single injections of 1 to 5 IU1,6,12,16. Alternatively, a dose of 4 IU/kg body weight can be used12. A single dose of 2 IU can be repeated once if difficulty milking is encountered.</li>
	<li>Inject the oxytocin intraperitoneally. Insert the needle into the lower right quadrant of the abdomen with the needle pointing towards the head, at an angle of 15-30&#176;, about 0.5 cm deep.</li>
	<li>Pull back on the plunger to ensure negative pressure prior to injection. If any fluid (blood, urine, intestinal contents, etc.) is aspirated into the syringe, remove the needle and attempt the injection with a new needle and syringe. If no fluid is aspirated inject the oxytocin and discard the needle and syringe immediately into a biohazard container.</li>
	<li>Wait approximately 5-15 min for the oxytocin to stimulate milk letdown.</li>
</ol>
5. Preparation of Milking Sites
<ol>
	<li>Choose the sites/teats from which milk will be collected. Milk can be collected from any teat12.</li>
	<li>Gently remove the fur around the teats to be milked with the trimmers, as fur may cause difficulty in sample collection due to wicking of the milk. Be gentle - the skin around the teats is extremely sensitive and may be dry and as such is susceptible to scratches and tears.</li>
	<li>Sterilization of the teat is not necessary, but optionally, clean the teat with lukewarm water after the fur is removed. Prepare at least two sites as more than one site may be required for milk collection. 
	NOTE: If the milk analysis includes microbial profiling, the teat area may require sterilization with iodine5.</li>
</ol>
6. Milk Collection
<ol>
	<li>Gently squeeze the base of the teat, manually expelling the milk for collection. 
	NOTE: If the milk analysis includes microbial profiling, the first few drops of milk should be discarded.</li>
	<li>Collect the milk droplets into a capillary tube, filling the capillary tube. Capillary tubes that accommodate larger volumes (e.g., 50 &#181;l) ease the process. 
	NOTE: If difficulty in collecting milk is encountered, a second dose of oxytocin can be administered. It is recommended the dose not exceed 4 IU total.</li>
	<li>Dispense the milk from the capillary tube into a sterile microcentrifuge tube by touching the end of the tube that was used to draw milk from the teat to the side of the microcentrifuge tube - observe the milk being drawn out of the capillary tube via capillary action. 
	NOTE: &#39;Blow out&#39; the milk that is not drawn into the tube using an 18 G needle attached to a 1 ml syringe.</li>
	<li>Continually monitor the dam for signs of pain or respiratory depression and adjust the flow of isoflurane accordingly.</li>
	<li>Continue to collect milk as described in this section until sufficient milk has been collected for the chosen milk analysis. For the creamatocrit and protein concentration determination described below, collect 0.25 ml of milk. 
	NOTE: Use a different milking site if milk letdown slows or the chosen site does not expel milk sufficiently. A maximum of approximately 2.5 ml of milk per animal can be collected17, or up to 0.5 ml per teat. The authors recommend that total time under anesthetic be limited to approximately 45-60 min, or 45 min of milking.</li>
	<li>Collect milk into a microhematocrit tube for creamatocrit measurement from fresh milk samples, and seal the end of the tube with clay sealant. Label the microhematocrit tube with the dam ID and store the milk sample upright.</li>
	<li>When milking is complete, turn off the flow of isoflurane and oxygen. Remove the mask from the dam and continue to monitor dam until awake.&#160;It is recommended that if not fully conscious, the dam should be placed on an absorbent bench pad during the recovery period, rather than directly on the cage bedding, to prevent the bedding from being aspirated or scratching the dam&#8217;s eyes during recovery.&#160; 
	NOTE: Do not leave the dam unattended until it has regained sufficient consciousness to maintain sternal recumbancy.</li>
	<li>If no further analyses are required, freeze milk at -80 &#176;C. Others have suggested that milk can be stored for up to 3 hr at 4 &#176;C or 5 months at -20 &#176;C18.</li>
</ol>
7. Creamatocrit Measurement
<ol>
	<li>Estimate the fat content in the milk by calculating the milk creamatocrit (the percentage of cream in the milk sample)19. 
	NOTE: Measurements with human milk have demonstrated that either fresh or frozen milk can be used for a creamatocrit measurement, however fresh milk is more highly correlated (r = 0.92 versus r = 0.90) with lipid concentration11. The use of fresh or frozen milk for creamatocrit measurements should be kept consistent across the study, as thawed milk is associated with a small decrease in creamatocrit values11.</li>
	<li>For fresh milk, collect a sample of milk from the teat into a microhematocrit tube; fill at least &#190; full (approximately 15-20 &#181;l). Alternatively, draw fresh milk from the collected sample into the capillary tube after mixing well. Seal the end with clay sealant.</li>
	<li>Place the capillary tube into the hematocrit spinner, with the sealed end pointing towards the outside, ensuring the centrifuge is balanced.</li>
	<li>Begin the hematocrit spin (120 sec at 13,700 x g). 
	NOTE: Spin time or speed may change depending on the model of centrifuge used.</li>
	<li>Remove the tube from the centrifuge after the spin is complete and perform the measurements for calculating the creamatocrit. Observe the sample&#39;s separation into a cream layer and a clear layer.</li>
	<li>Measure and record the total length of fluid in the tube and the length of the fat (cream) layer using calipers or a ruler. 
	NOTE: The creamatocrit is expressed as the percentage of the cream layer within the the milk sample19, calculated as (length of cream layer/total length of milk column) x 100 (Figure 1A). Calculate fat concentration and energy values from the creamatocrit measurement as follows: Fat concentration (g/L) = (creamatocrit (%)-0.59)/0.146 (Figure 1B)19; Energy value (kcal/L) = 290 + (66.8*creamatocrit (%) (Figure 1C)19.</li>
</ol>
8. Protein Concentration Determination
<ol>
	<li>Using Bovine Serum Albumin as a protein standard, determine total milk protein concentration using a standard protein assay, such as a Lowry protein assay6. 
	NOTE: Dilution of the milk may be necessary for the milk protein measurements to fall within the standard curve of the assay.</li>
</ol>

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The authors have no conflicts of interest to disclose. All animal experiments were conducted in accordance with CCAC approved protocols.

Investigations into maternal milk components have increased as interest in early life development research rises. As the sole source of nutrition during the neonatal period, the bioactive compounds in milk are essential for ideal growth and development, especially in the context of intestinal and immune health3. The method presented here is a simple, non-invasive method of collecting milk from the lactating rat in amounts sufficient for downstream analysis, such as oligosaccharide profiling6. The me...

Milk is the sole source of nutrition for newborn mammals, providing energy and nutrients for infant growth and development1,2. While milk mainly consists of cells, lipids, and protein1, it also contains a plethora of bioactive compounds that modulate early life development of offspring including enzymes, carbohydrates, hormones, antibodies, growth factors, cytokines, exosomes, microvesicles, and small RNAs such as microRNA1,2. The fundamental role of maternal milk in the establishment of offspring immune and intestinal health3, coupled with evidence that breastfed infants are less susceptible to disease2, highlights the importance of identifying the milk constituents associated with disease processes in early life and the molecular mechanisms involved in their actions. The developing rat is a popular model for investigating the effect of various nutritional, physiological, and chemical interventions on early-life development4. The analysis of rat milk may therefore provide novel insight into maternal and offspring health.
Current scientific advances now provide increasing opportunities for in-depth investigations of the effects of specific milk constituents on health and disease. For example, sequencing of milk bacterial profiles has elucidated their role in early intestinal colonization of the infant gut5, mass spectrometry analysis of milk oligosaccharides have provided insight into the alteration of milk oligosaccharide profiles via maternal diet6, and deep sequencing of microRNA secreted in the fat globules of breast milk highlights possible roles in gene transcription, metabolism, and immune function7.
Rat models represent one of the most popular model organisms used in maternal studies8,9. One advantage is their short gestation and lactation periods, lasting only approximately 21 days each; therefore the total time from the start of pregnancy to lactation represents a short period of time in which valuable data can be generated. The larger size of rats compared to mice, in the context of milk collection, may provide a significant advantage with respect to volume of milk and ease of milk collection; milk production in the mouse, for example, seems to be dependent on total body weight with heavier mice producing more milk10.
Here, a general description for the manual collection of milk from lactating rats is provided. This protocol requires minimal equipment, is non-invasive, inexpensive, and can be used to collect adequate volumes of milk for further downstream analyses. In brief, the dam is anesthetized with isoflurane, milk letdown is stimulated by oxytocin, and milk is collected into capillary tubes via manual expression of the milk. Finally, as two major components of milk are fat and proteins, a brief description of estimating milk fat content using creamatocrit measurements11 and quantification of total protein concentration using a standard protein assay is presented.

<table><tbody><tr><td>&lt;strong&gt;&lt;em&gt;Equipment - Milking&lt;/em&gt;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>1 ml syringes</td><td>BD-Canada</td><td>309602</td><td></td></tr><tr><td>25 G needles</td><td>BD-Canada</td><td>305122</td><td></td></tr><tr><td>18 G needles</td><td>BD-Canada</td><td>305196</td><td></td></tr><tr><td>50 &amp;mu;l Microdispenser Capillary Tubes</td><td>Fisher Scientific</td><td>21-169D</td><td></td></tr><tr><td>Oxytocin (20 USP Units/ml)</td><td>Bimeda-MTC</td><td>1OXY015</td><td></td></tr><tr><td>PPC Vet Isoflurane Inhalation Anesthetic, 250 ml</td><td>Fresenius Kabi</td><td>M60302</td><td>Used on the order of a veterinarian</td></tr><tr><td>Sterile Alcohol Prep Pad</td><td>Dukal</td><td>853</td><td></td></tr><tr><td>Absorbent Bench Underpad</td><td>VWR</td><td>82020-845</td><td></td></tr><tr><td>Maxi-Therm Hyper/Hypothermia Blanket</td><td>Cincinnati Sub-Zero</td><td>274</td><td></td></tr><tr><td>Rodent Anesthesia Machine with Vaporizer</td><td>Benson Medical Industries Inc.</td><td></td><td>Subject to individual laboratory needs</td></tr><tr><td>Animal Masks</td><td>Benson Medical Industries Inc.</td><td>50100/50102</td><td></td></tr><tr><td>Microcentrifuge Tubes</td><td>Axygen</td><td>MCT-060-C</td><td></td></tr><tr><td>ChroMini Professional Trimmer</td><td>Wahl</td><td></td><td></td></tr><tr><td>&lt;strong&gt;&lt;em&gt;Equipment - Creamatocrit&lt;/em&gt;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>StatSpin SafeCrit Plastic Microhematocrit Tubes (Untreated)</td><td>Fisher Scientific</td><td>22-274-914</td><td></td></tr><tr><td>Critoseal Capillary Tube Sealant Tray</td><td>VWR</td><td>470161-478</td><td></td></tr><tr><td>StatSpin CritSpin Microhematocrit Centrifuge</td><td>Beckman Coulter, Inc</td><td>X00-004999-001</td><td></td></tr></tbody></table>

milk collection in the rat using capillary tubes and estimation of milk fat content by creamatocrit

Milk, as the sole source of nutrition for the newborn mammal, provides the necessary nutrients and energy for offspring growth and development. It also contains a vast number of bioactive compounds that greatly affect the development of the neonate. The analysis of milk components will help elucidate key factors that link maternal metabolism and health with offspring growth and development. The laboratory rat represents a popular model organism for maternal studies, and rat milk can be used to examine the effect of various maternal physiological, nutritional, and pharmacological interventions on milk components, which may then impact offspring health. Here a simple method of manually collecting milk from the lactating rat that can be performed by a single investigator, does not require specialized vacuum or suction equipment, and provides sufficient milk for subsequent downstream analysis is described. A method for estimating the fat content of milk by measuring the percentage of cream within the milk sample, known as the creamatocrit, is also presented. These methods can ultimately be used to increase insight into maternal-child health and to elucidate maternal factors that are involved in proper growth and development of offspring.

Milk was collected as described at weaning from Wistar dams (approximately 22 weeks old, weighing 350 to 400 g) that consumed a control (AIN-93G, n = 5), high protein (40% casein wt/wt, n = 5), or high prebiotic fibre (21.6% wt/wt, 1:1 ratio of oligofructose and inulin, n = 4) diet throughout pregnancy and lactation. The oxytocin dose was 2 IU. Milk was collected using capillary tubes, and one tube was spun using a hematocrit spinner to determine creamatocrit (Figure 1A), which was then used to estimate ...

Watch this Scientific Journal Video about Milk Collection in the Rat Using Capillary Tubes and Estimation of Milk Fat Content by Creamatocrit at JoVE.com

Milk Collection in the Rat Using Capillary Tubes and Estimation of Milk Fat Content by Creamatocrit

Milk is a primary source of nutrition for the neonate. Analysis of milk components may provide insight into maternal factors that affect offspring health. This protocol describes a manual method of collecting milk samples from the lactating rat, which can then be used for further downstream analysis.

Milk, as the sole source of nutrition for the newborn mammal, provides the necessary nutrients and energy for offspring growth and development. It also ...

milk-collection-rat-using-capillary-tubes-estimation-milk-fat-content

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JoVE Journal

Biology

12.3K Views.  University of Calgary. Milk is a primary source of nutrition for the neonate. Analysis of milk components may provide insight into maternal factors that affect offspring health. This protocol describes a manual method of collecting milk samples from the lactating rat, which can then be used for further downstream analysis.

Video: Milk Collection in the Rat Using Capillary Tubes and Estimation of Milk Fat Content by Creamatocrit

The Use of Gas Chromatography to Analyze Compositional Changes of Fatty Acids in Rat Liver Tissue during Pregnancy

Gas chromatography (GC) is a highly sensitive method used to identify and quantify the fatty acid content of lipids from tissues, cells, and plasma/serum, yielding results with high accuracy and high reproducibility. In metabolic and nutrition studies GC allows assessment of changes in fatty acid concentrations following interventions or during changes in physiological state such as pregnancy. Solid phase extraction (SPE) using aminopropyl silica cartridges allows separation of the major lipid classes including triacylglycerols, different phospholipids, and cholesteryl esters (CE). GC combined with SPE was used to analyze the changes in fatty acid composition of the CE fraction in the livers of virgin and pregnant rats that had been fed various high and low fat diets. There are significant diet/pregnancy interaction effects upon the omega-3 and omega-6 fatty acid content of liver CE, indicating that pregnant females have a different response to dietary manipulation than is seen among virgin females.

Pregnancy leads to significant changes to the fatty acid composition of maternal tissues. Lipid profiles can be obtained via gas chromatography to allow identification and quantification of fatty acids in individual lipid classes among rats fed various high and low fat diets during pregnancy.

Gas chromatography (GC) is a highly sensitive method used to identify and quantify the fatty acid content of lipids from tissues, cells, and plasma/serum, ...

Chemistry

Rat Mammary Epithelial Cell Transplantation into the Interscapular White Fat Pad

As early as the 1970s, researchers have successfully transplanted mammary epithelial cells into the interscapular white fat pad of rats. Grafting mammary epithelium using transplantation techniques takes advantage of the hormonal environment provided by the adolescent rodent host. These studies are ideally suited to explore the impact of various biological manipulations on mammary gland development and dissect many aspects of mammary gland biology. A common, but limiting, feature is that transplanted epithelial cells are strongly influenced by the surrounding stroma and outcompeted by endogenous epithelium; to utilize native mammary tissue, the abdominal-inguinal white fat pad must be cleared to remove host mammary epithelium prior to the transplantation. A major obstacle when using the rat model organism is that clearing the developing mammary tree in post-weaned rats is not efficient. When transplanted into gland-free fat pads, donor epithelial cells can repopulate the cleared host fat pad and form a functional mammary gland. The interscapular fat pad is an alternative location for these grafts. A major advantage is that it lacks ductal structures yet provides the normal stroma that is necessary to promote epithelial outgrowth and is easily accessible in the rat. Another major advantage of this technique is that it is minimally invasive, because it eliminates the need to cauterize and remove the growing endogenous mammary tree. Additionally, the interscapular fat pad contains a medial blood vessel that can be used to separate sites for grafting. Because the endogenous glands remain intact, this technique can also be used for studies comparing the endogenous mammary gland to the transplanted gland. This paper describes the method of mammary epithelial cell transplantation into the interscapular white fat pad of rats.

This article describes a transplantation method to graft donor rat mammary epithelial cells into the interscapular white fat pad of recipient animals. This method can be used to examine host and/or donor effects on mammary epithelium development and eliminates the need for pre-clearing, thereby extending the usefulness of this technique.

As early as the 1970s, researchers have successfully transplanted mammary epithelial cells into the interscapular white fat pad of rats. Grafting mammary ...

Cancer Research

Prevention of Heat Stress Adverse Effects in Rats by Bacillus subtilis Strain

This study was designed to evaluate the protective effect of the Bacillus subtilis strain against complications related to heat stress. Thirty-two Sprague-Dawley rats were used in this study. Animals were orally treated twice a day for two days with B. subtilis BSB3 strain or PBS. The next day after the last treatment, each group was divided and two experimental groups (one treated with PBS and one treated with B. subtilis) were placed at 45 oC for 25 min. Two control groups stayed for 25 min at room temperature. All rats were euthanized and different parameters were analyzed in all groups. Adverse effects of heat stress are registered by the decrease of villi height and total mucosal thickness in the intestinal epithelium; translocation of bacteria from the lumen; increased vesiculation of erythrocytes and elevation of the lipopolysaccharides (LPS) level in the blood. The protective efficacy of treatment is evaluated by prevention of these side effects. The protocol was set up for the oral treatment of rats with bacteria for prevention of heat stress complications, but this protocol can be modified and used for other routes of administration and for analysis of different compounds.

Prevention of Heat Stress Adverse Effects in Rats by Bacillus subtilis Strain

We described a protocol for prevention of heat stress effects in rats by oral pre-treatment with beneficial bacteria. This protocol can be modified and used for various routes of administration and for analysis of different compounds.

This study was designed to evaluate the protective effect of the Bacillus subtilis strain against complications related to heat stress. Thirty-two ...

Immunology and Infection

Repeated Blood Collection from Tail Vein of Non-Anesthetized Rats with a Vacuum Blood Collection System

Blood can be collected from rats in a number of sampling locations. For instance, the tail vein is a superior location for blood sampling. However, the tail vein is thin so that it is sometimes hard to puncture. In addition, the tail vein has low blood flow and requires a long sampling time to get sufficient blood. The present report describes a simple blood sampling method, the vacuum blood collection method, which is usually used to obtain blood samples from patients, here used for non-anesthetized rats. The 22 G butterfly needle tip was inserted into one of the lateral tail veins approximately 2-3 cm from the tip of the tail at an angle of approximately 20&#176;, and blood was collected in the vacuum collection tube by inserting the rubber end of the butterfly needle into the vacuum blood collection tube. The present experimental results show that the success rate was 95% in the experimental group and 90% in the beginner group. The success rate and puncture times were similar between two groups. The sampling duration was significantly shorter in the experimental group compared to beginner group. In conclusion, this vacuum blood collection method for sequential blood sampling from the tail vein of non- anesthetized rats is feasible and easy-to-learn, which might serve as a reliable alternative to other conventionally used blood sampling techniques for rats.

Here, we describe a simple tail vein blood sampling method in non-anesthetized rats using a vacuum extraction tube system. This method reduces the risk of direct exposure to blood and simplifies taking multiple samples from a single venipuncture.

Blood can be collected from rats in a number of sampling locations. For instance, the tail vein is a superior location for blood sampling. However, the ...

Medicine

Three-Dimensional Culture of Vascularized Thermogenic Adipose Tissue from Microvascular Fragments

Engineering thermogenic adipose tissue (e.g., beige or brown adipose tissues) has been investigated as a potential therapy for metabolic diseases or for the design of personalized microtissues for health screening and drug testing. Current strategies are often quite complex and fail to accurately fully depict the multicellular and functional properties of thermogenic adipose tissue. Microvascular fragments, small intact microvessels comprised of arteriole, venules, and capillaries isolated from adipose tissue, serve as a single autologous source of cells that enable vascularization and adipose tissue formation. This article describes methods for optimizing culture conditions to enable the generation of three-dimensional, vascularized, and functional thermogenic adipose tissues from microvascular fragments, including protocols for isolating microvascular fragments from adipose tissue and culture conditions. Additionally, best practices are discussed, as are techniques for characterizing the engineered tissues, and sample results from both rodent and human microvascular fragments are provided. This approach has the potential to be utilized for the understanding and development of treatments for obesity and metabolic disease.

Here, we present a detailed protocol outlining the use of microvascular fragments isolated from rodent or human fat tissue as a straightforward approach to engineer functional, vascularized beige adipose tissue.

Engineering thermogenic adipose tissue (e.g., beige or brown adipose tissues) has been investigated as a potential therapy for metabolic diseases or for ...

Bioengineering

Intranasal Immunization and Milk Collection in Studies of Maternal Immunization in New Zealand White Rabbits (Oryctolagus cuniculus)

Due to similarities in placentation and antibody transfer with humans, rabbits are an excellent model of maternal immunization. Additional advantages of this research model are the ease of breeding and sample collection, relatively short gestation period, and large litter sizes. Commonly assessed routes of immunization include subcutaneous, intramuscular, intranasal, and intradermal. Nonterminal sample collection for the chronological detection of the immunologic responses to these immunizations include the collection of blood, from both dams and kits, and milk from the lactating does. In this article, we will demonstrate techniques our lab has utilized in studies of maternal immunization in New Zealand White rabbits (Oryctolagus cuniculus), including intranasal immunization and milk collection.

Intranasal Immunization and Milk Collection in Studies of Maternal Immunization in New Zealand White Rabbits Oryctolagus cuniculus

This article describes and demonstrates the administration of intranasal vaccines and the collection of milk from lactating rabbits (Oryctolagus cuniculus) as a means to assess mucosal immunity in a translationally appropriate model of maternal immunization.

Due to similarities in placentation and antibody transfer with humans, rabbits are an excellent model of maternal immunization. Additional advantages of ...

Milk Collection Methods for Mice and Reeves' Muntjac Deer

Animal models are commonly used throughout research laboratories to accomplish what would normally be considered impractical in a pathogen&#8217;s native host. Milk collection from animals allows scientists the opportunity to study many aspects of reproduction including vertical transmission, passive immunity, mammary gland biology, and lactation. Obtaining adequate volumes of milk for these studies is a challenging task, especially from small animal models. Here we illustrate an inexpensive and facile method for milk collection in mice and Reeves&#8217; muntjac deer that does not require specialized equipment or extensive training. This particular method requires two researchers: one to express the milk and to stabilize the animal, and one to collect the milk in an appropriate container from either a Muntjac or mouse model. The mouse model also requires the use of a P-200 pipetman and corresponding pipette tips. While this method is low cost and relatively easy to perform, researchers should be advised that anesthetizing the animal is required for optimal milk collection.

Milk collection from animal models facilitates various research avenues: understanding passive immunity, identifying pathogens responsible for vertical transmission and, through the use of transgenic mice, even commercial production of proteins found in human breast milk. Here we illustrate a simple method for milk collection in mice and Reeves&#8217; muntjac deer.

Animal models are commonly used throughout research laboratories to accomplish what would normally be considered impractical in a pathogen&#8217;s native ...

Preparation of Rat Serum Suitable for Mammalian Whole Embryo Culture

Mammalian whole embryo culture (WEC) is a widely used technique for examining pharmacological toxicity in developing mouse and rat embryos and for investigating the mechanisms of developmental processes. Immediately centrifuged (IC) rat serum is commonly used for WEC and is essential for the growth and development of cultured mouse and rat embryos ex vivo. For the culture of midgestation embryos (i.e., E8.0-12.5 for the mouse, and E10.0-14.5 for the rat), 100% rat serum is the best media for supporting the growth of the embryo ex vivo. To prepare rat serum suitable for WEC, the collected blood should be centrifuged immediately to separate the blood cells from the plasma fraction. After centrifugation, the fibrin clot forms in the upper layer; this clot should be squeezed gently using a pair of sterile forceps and subsequently centrifuged to completely separate the blood cells from the serum. In this video article, we demonstrate our standard protocol for the preparation of optimal IC rat serum, including blood collection from the abdominal aorta of male rats and extraction of the serum by centrifugation.

Mammalian whole embryo culture (WEC) is widely used in teratology and developmental biology. Immediately centrifuged rat serum is commonly provided as a medium for both mouse and rat WEC. In this video, we demonstrate our standard protocol for the preparation of high-quality rat serum suitable for mammalian WEC.

Mammalian whole embryo culture (WEC) is a widely used technique for examining pharmacological toxicity in developing mouse and rat embryos and for ...

Cerebrospinal Fluid and Blood Withdrawal from Rats with Concurrent EEG Recording

Sampling Cerebrospinal Fluid and Blood from Lateral Tail Vein in Rats During EEG Recordings

Because the composition of body fluids reflects many physiological and pathological dynamics, biological liquid samples are commonly obtained in many experimental contexts to measure molecules of interest, such as hormones, growth factors, proteins, or small non-coding RNAs. A specific example is the sampling of biological liquids in the research of biomarkers for epilepsy. In these studies, it is desirable to compare the levels of molecules in cerebrospinal fluid (CSF) and in plasma, by withdrawing CSF and plasma in parallel and considering the time distance of the sampling from and to seizures. The combined CSF and plasma sampling, coupled with video-EEG monitoring in epileptic animals, is a promising approach for the validation of putative diagnostic and prognostic biomarkers. Here, a procedure of combined CSF withdrawal from cisterna magna and blood sampling from the lateral tail vein in epileptic rats that are continuously video-EEG monitored is described. This procedure offers significant advantages over other commonly used techniques. It permits rapid sampling with minimal pain or invasiveness, and reduced time of anesthesia. Additionally, it can be used to obtain CSF and plasma samples in both tethered and telemetry EEG recorded rats, and it may be used repeatedly across multiple days of experiment. By minimizing the stress due to sampling by shortening isoflurane anesthesia, measures are expected to reflect more accurately the true levels of investigated molecules in biofluids. Depending on the availability of an appropriate analytical assay, this technique may be used to measure the levels of multiple, different molecules while performing EEG recording at the same time.

Author Spotlight: Obtaining High-Quality CSF and Blood Samples for Epilepsy Biomarker Discovery 

The protocol shows repeated cerebrospinal fluid and blood collections from epileptic rats performed in parallel with continuous video-electroencephalogram (EEG) monitoring. These are instrumental for exploring possible links between changes in various body fluid molecules and seizure activity.

Because the composition of body fluids reflects many physiological and pathological dynamics, biological liquid samples are commonly obtained in many ...

Neuroscience

Subclavian Vein Puncture Method for Repeated Blood Collection in Conscious Rats

Subclavian Vein Blood Sampling in Conscious Rats

There are several established methods for obtaining repeated blood samples from rats, with the most commonly employed methods being lateral tail vein sampling without anesthesia and jugular vein sampling with anesthesia. However, most of these methods require assistance and anesthetic equipment and sometimes pose difficulties in terms of blood collection or the poor quality of blood samples. In addition, these methods of blood collection consume significant time and human resources when repeated blood sampling is required for a large number of rats. This study presents a technique for repetitive blood sampling in non-anesthetized rats by a single proficient individual. Highly satisfactory blood samples can be obtained by puncturing the subclavian vein. The method demonstrated an impressive overall success rate of 95%, with a median time of merely 2 min from rat restraint to the completion of blood collection. Furthermore, performing consecutive blood collections within the designated range does not inflict any harm on the rats. This method is worth promoting for blood collection, especially in large-scale pharmacokinetic studies.

Author Spotlight: Developing an Efficient Blood Collection Method in Non-Anesthetized Rats 

Here we present a combination of effective rat restriction and subclavian vein puncture methods that enable rapid, safe, and repeated blood collection in rats without anesthesia.

There are several established methods for obtaining repeated blood samples from rats, with the most commonly employed methods being lateral tail vein ...

Milk Collection in the Rat Using Capillary Tubes and Estimation of Milk Fat Content by Creamatocrit

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The Use of Gas Chromatography to Analyze Compositional Changes of Fatty Acids in Rat Liver Tissue during Pregnancy

Rat Mammary Epithelial Cell Transplantation into the Interscapular White Fat Pad

Prevention of Heat Stress Adverse Effects in Rats by Bacillus subtilis Strain

Repeated Blood Collection from Tail Vein of Non-Anesthetized Rats with a Vacuum Blood Collection System

Three-Dimensional Culture of Vascularized Thermogenic Adipose Tissue from Microvascular Fragments

Intranasal Immunization and Milk Collection in Studies of Maternal Immunization in New Zealand White Rabbits (Oryctolagus cuniculus)

Milk Collection Methods for Mice and Reeves' Muntjac Deer

Preparation of Rat Serum Suitable for Mammalian Whole Embryo Culture

Sampling Cerebrospinal Fluid and Blood from Lateral Tail Vein in Rats During EEG Recordings

Subclavian Vein Blood Sampling in Conscious Rats